Antibodies and derivatives thereof are used in many areas of human and veterinary therapy, diagnostics and monitoring. One problem of utilizing the naturally occurring antibodies is the preparation thereof. The antibodies are still produced in an animal cell culture system, which is a very costly method. In some applications such as, for example, preparation of fusion proteins or a therapeutic use which requires rapid blood clearance and good tissue penetration, the size of naturally occurring antibody molecules represents another problem (Colcher et al., 1998). Recombinant antibody molecules such as scFvs (Bird et al., 1988), Miniantibodies (Pack and Plückthun, 1992) or bispecific antibodies (Holliger and Winter, 1993) are mainly composed of just the antigen-binding domains of the antibodies (VH and VL). Owing to their considerably reduced size, they show improved tissue penetration and are also better suited for fusions with other proteins than complete antibodies. Compared with the latter, though, recombinant antibody fragments are often more unstable, have low affinities and are difficult to prepare in recombinant form, owing to the disulphide bridges to be formed. Methods for stabilization and improved affinity of the recombinant antibody fragments include, inter alia testing various linker peptides and introduction of disulphide linkages (Glockshuber et al., 1990, Cumber et al., 1992, Brinkmann, 1997).
The sequence and length of the linker peptides can influence both the stability against proteases and the affinity of the antibody fragments (Pantoliano et al., 1991). The introduction of additional disulphide linkages into the conserved framework regions of the variable domains can lead to increased resistance to heat (Young et al., 1995) and denaturing agents and to increased yields in heterologous expression. In general, however, many scFvs show low stability and tend to aggregate already at 37° C. The instability may also be caused by using the common Fv-fragment cloning primers which can introduce new destabilizing mutations. The antibody fragments are produced in the bacterial system mainly by exporting into the periplasmic space, and optimizations regarding the redox state and simultaneous expression of foldings helpers are possible here, too.